The present invention discloses the use of a mutant Leishmania as a suicidal vaccine wherein the mutant Leishmania is responsive to external signals to become porphyric and commit suicidal cytolysis.
Leishmania spp. are flagellated trypanosomatid protozoa commonly known as trypanosomes. Essentially all of them are parasites, of which overwhelming majority are non-pathogenic to humans, but live in other animals and sometimes in plants. However, certain species of Leishmania are pathogenic to their hosts, resulting in leishmaniasis. The best known pathogenic species are vector-borne Trypanosoma spp., which cause African Sleeping Sickness as extracellular parasites in body fluids and South American Chagas disease as parasites with both extracellular and intracellular phases. Recently, Leishmania spp. have attracted attention for causing “Baghdad boils” via sand fly bites among soldiers sent to Iraq and Afghanistan.
Leishmania spp. are uniquely suitable among trypanosomes to serve as a live vaccine model for the following reasons:
First, Leishmania spp. naturally infect antigen-presenting cells (APC), i.e., dendritic cells and macrophages, hence inherently suitable for homing vaccines to a desired destination. Leishmania not only infect macrophages but utilize them as their exclusive host cells by residing in their phagolysosomes (Chang and Dwyer, 1976). This is the very site where antigens/vaccines are processed for presentation via the lysosomal major histocompatibility complex (MHC) Class II pathway and also via the cytosolic MHC Class I pathway by cross-presentation (Houde et al. 2003). This APC-homing ability of Leishmania makes them exceptionally attractive for consideration as a live vaccine model. Micro-organisms, which share with Leishmania this specific feature, are very few and limited to pathogenic bacteria, e.g., Coxiella burnetii, Legionella pneumophilia and Mycobacterium leprae. Unlike these microbes, Leishmania have a number of advantages as single-cell eukaryotes.
Second, Leishmania spp. are amenable to laboratory maintenance in culture and in animal models. They grow in vitro as flagellated motile “promastigotes”, which normally live as extracellular parasites in the gut of blood sucking sand flies. Continuous cultivation of promastigotes is readily achievable for most species by using tissue culture media commercially available for mammalian and insect cells (Chang and Hendricks, 1985). These promastigotes differentiate into infective or “metacyclic” forms in fly gut or as they reach stationary phase in culture. They further differentiate into non-motile “amastigotes”—the intra-macrophage stage normally found in the mammalian hosts. Some species, especially L. amazonensis and L. mexicana, grow readily and continuously free of macrophages as “axenic amastigotes” under suitable conditions at acidic pH and mammalian body temperature (Hodgkinson et al., 1996). These species also grow continuously as intracellular amastigotes in macrophage cell lines, e.g., J774 series (Chang, 1980; Chang, 1978; Chang and Hendricks, 1985). These and other species can be further passaged cutaneously or viscerally in rodents, e.g., hamsters and mice (Chang and Hendricks, 1985).
Third, non-pathogenic Leishmania spp. exist, originating from lizards, e.g., L. tarentolae and from rodents, e.g., L. enriettii of guinea pig and L. turanica of the great gerbil in Gobi desert. There is documented evidence for these species to produce no disease in human volunteers at least for L. gerbilli (Strelkova, 1991), despite it is phylogenetically closely related to L. major (FIG. 1). L. major is well-known to cause self-healing simple cutaneous leishmaniasis (CL). This is the least pathogenic one among 2 dozens or so additional species of known human pathogens in both Leishmania and Viannia subgenus (Shaw, 1994). Some of them cause not only CL but also facial disfiguring mucocutaneous leishmaniasis (MCL), e.g., L. braziliensis and L. panamensis or the potentially fatal visceral leishmaniasis (VL), e.g., the Indian kala-azar caused by L. donovani and the Mediterranean infantile VL (IVL) caused by L. infantum. Included for the present invention are well-established laboratory species as well as field isolates appropriately genotyped (see legend to FIG. 1), ranging from totally non-pathogenic to highly virulent species.
Fourth, human populations have been successfully vaccinated against cutaneous leishmaniasis with lesion-derived live amastigotes of L. major. This is known as a time-honored procedure of “Leishmanization” (i.e., vaccination of children with virulent parasites from diseased patients). It has been practiced for centuries and proven effective to elicit life-long protective immunity against CL in the Middle East and Central Asia. Iranian and Russian workers have documented this more scientifically. From 1978 to 1989, Mohebali et al. had “leishmanized” or vaccinated about 2 million individuals in their upper arms with a live L. major culture originally from a Great Gerbil in Iran. This “vaccination” confers protective immunity in >90% of the leishmanized individuals, but the procedure takes 10-15 months, during which period “vaccinees” develop full-blown lesions followed by spontaneous healing, just like the natural infection (Nadim et al., 1997). In addition, non-healing lesions occurred in 1-2/10,000 cases. Similar results were reported in Central Asia using a different strain of live L. major by Dr. Leonid Krasnonos et al. in Isaev Institute of Medical Parasitology, Samakan, Uzbekistan. Thus, “Leishmanization” has a proven track record of effectiveness in contrast to the uncertain benefits of BCG vaccination for tuberculosis. Leishmania also compare favorably over BCG to serve as potential vehicles to deliver add-on vaccines, since these and other related trypanosomes are readily cultivable and since they are genetically efficient and versatile to express foreign genes. One principal disadvantage common to all live vaccine models is the issue of residual pathogenicity, as described for “Leishmanization”. An important aspect of the present disclosure is to address this issue by using transgenic Leishmania responsive to external signals to commit suicidal cytolysis.
Fifth, there is a substantial body of information available for the immune response of animals to Leishmania infection. Experimental leishmaniasis has been extensively used for elucidating rodent immunogenetics and immunobiology, especially the BALB/c mouse-L. major model (Gumy et al., 2004). The use of this model has indeed led to the significant discovery in the dichotomy of animal immune response via Th1 and Th2 pathways (Locksley et al., 1987; Scott et al., 1988). The regulation of these pathways in relation to protective immunity is still under intensive investigation (Etges and Muller, 1998), including the use of different Leishmania-mouse combinations (Lipoldova et al., 2002). There have been additional efforts to extend such investigation to hamster and primate models (Melby et al., 2001; Olobo et al., 2001; Probst et al., 2001; Requena et al., 2000). This wealth of knowledge has proven its value in current efforts to develop Leishmania DNA-based and recombinant peptide vaccines as well as using sand fly saliva antigens (Ahmed et al., 2004; Belkaid et al., 1996; Belkaid et al. 1998; Campbell et al., 2003; Campbell et al., 2004; Coler et al., 2002; Kamhawi et al., 2000; Morris et al.,; 2001; Reed, 2001; Valenzuela et al., 2001).
Sixth, Leishmania spp. are amenable to genetic manipulations for expressing endogenous or foreign genes via transfection by electroporation. The genome project has been essentially completed for L. major. This and other species possess all the necessary machineries of eukaryotic types not only for gene replication, mRNA transcription and protein translation but also for post-translational modifications of proteins. The last cellular events, which may have some importance in vaccine presentation, are absent in bacterium-based live vaccine models, e.g., Salmonella and BCG. There are at least 6 different markers available for selection of transfectants in conjunction with the use of Leishmania-E. coli shuttle vectors. The principal is the pX series (Freedman and Beverley, 1993; Goyard and Beverley, 2000). In addition, we have developed p6.5 using Leishmania endogenous N-acetylglucosamine-1-phosphate transferase gene (nagt) as a selectable marker for tunicamycin (TM)-resistance (Chen et al., 2000, Kawazu et al., 1997; Liu and Chang, 1992; Sah et al., 2002; Somanna et al., 2002). There have been numerous reports of Leishmania gene knock-outs and knock-ins by homologous recombination (Alexander et al., 1998; Burchmore et al., 2003; Ilg et al., 2001; Cruz et al., 1991; Spath et al., 2003, 2004; Zhang et al., 2003). Such genetic manipulations have been routinely carried out by us (Chen et al., 2000; Chen et al., 1999; Sah et al., 2002) and will be used for the proposed studies.
Seventh, vaccination of animals by genetically modified Leishmania has produced favorable outcome. Mice were protected against infection by L. major or L. mexicana when immunized with their respective knockouts of house-keeping genes, e.g., dhfr (Titus et al., 1995) or virulence genes, i.e., Ipg 1, lpg2 and cysp (Alexander et al., 1998; Ilg et al., 2001; Spath et al., 2003, 2004).